Benoît Delatour

Detection by voxel-wise statistical analysis of significant changes in regional cerebral glucose uptake in an APP/PS1 transgenic mouse model of Alzheimer's disease

Biomarkers and technologies similar to those used in humans are essential for the follow-up of Alzheimers disease (AD) animal models, particularly for the clarification of mechanisms and the screening and validation of new candidate treatments. In humans, changes in brain metabolism can be detected by 1-deoxy-2-[(18)F] fluoro-D-glucose PET (FDG-PET) and assessed in a user-independent manner with dedicated software, such as Statistical Parametric Mapping (SPM). FDG-PET can be carried out in small animals, but its resolution is low as compared to the size of rodent brain structures. In mouse models of AD, changes in cerebral glucose utilization are usually detected by [(14)C]-2-deoxyglucose (2DG) autoradiography, but this requires prior manual outlining of regions of interest (ROI) on selected sections. Here, we evaluate the feasibility of applying the SPM method to 3D autoradiographic data sets mapping brain metabolic activity in a transgenic mouse model of AD. We report the preliminary results obtained with 4 APP/PS1 (64+/-1 weeks) and 3 PS1 (65+/-2 weeks) mice. We also describe new procedures for the acquisition and use of blockface photographs and provide the first demonstration of their value for the 3D reconstruction and spatial normalization of post mortem mouse brain volumes. Despite this limited sample size, our results appear to be meaningful, consistent, and more comprehensive than findings from previously published studies based on conventional ROI-based methods. The establishment of statistical significance at the voxel level, rather than with a user-defined ROI, makes it possible to detect more reliably subtle differences in geometrically complex regions, such as the hippocampus. Our approach is generic and could be easily applied to other biomarkers and extended to other species and applications.

Detection of vascular alterations by in vivo magnetic resonance angiography and histology in APP/PS1 mouse model of Alzheimer’s disease

ObjectThe brain of patients with Alzheimer’s disease (AD) is characterized by the presence of amyloid plaques and neurofibrillary tangles. Vascular alterations such as amyloid angiopathy are also commonly reported in patients with AD and participate in mechanisms involved in disease onset and progression. Transgenic mouse models of AD have been engineered to evaluate the pathophysiology and new treatments of the disease. Our study evaluated vascular alterations in APPSweLon/PS1M146L mouse model of AD.Materials and methodsHistological analysis and in vivo magnetic resonance angiography protocols based on time of flight (TOF) and contrast-enhanced (CE) angiography were applied to evaluate cerebrovascular alterations.ResultsHistological analysis showed that cerebrovascular amyloid deposition starts by the same time as extracellular amyloid plaques. However, unlike plaques deposition, severity of cerebrovascular alterations is stabilized in older animals. Alteration of the middle cerebral artery was detected in old APPSweLon/PS1M146L mice with respect to adult ones by evaluating the severity of vessel voids and the reduction of vessel length on TOF- and CE-angiograms. Age-related alterations in control PS1 mice were only detected as a reduced vessel length on CE-angiograms.ConclusionThese results show that macroscopic vascular abnormalities are part of the pathological alterations developed by APPSweLon/PS1M146L mouse models of AD.

APPxPS1dE9 mice display neuronal dysfunction at an asymptomatic state of amyloidosis

Pin1 alters the cis-trans isomerization of phosphorylated Ser/Thr-Pro motifs inamyloidprecursor protein (APP) and tau, facilitating the formation of plaques and tangles inAD.Conversely, restoration of Pin1 restores normal structure and function of tau, implicating the importance of Pin1 in neuronal protein regulation. Synaptic plasticity allows for an activatedNMDAreceptor to induce hundreds ofphosphorylatedSer/Thr-Pro motifs in several hundredPSDproteins. In this study, we observed Pin1 associations with synaptic proteins at dendritic rafts and the postsynaptic density (PSD), compared pathological changes of Pin1 in AD patients against controls, and demonstrated the effect of loss of Pin1 activity on synaptic proteins and structure. Methods: Human cortices from 12 AD patients and 10 age-matched controls were prepared using a tissue extraction protocol, followed by SDSPAGE, Western and Dot blot analyses. W e used Pin1 -/-, Pin1 shRNA or Pin1 inhibitor to alter theactivity ofPin1in cultured neurons. Immunocytochemistry and immunoblot were used to visualize and analyze Pin1-related changes in Shank proteins and dendritic spines.Results:We found that Pin1 protein, a common pathological cause linked to the products of misfolding proteins Ab and hyperphosphorylated tau, colocalizes with NMDA receptors and Shank3 proteins. Pin1 levels are pathologically altered at the synapse in AD patients, and loss of Pin1 activity may alter the function of glutamate receptors and the levels of Shank proteins and dendritic spines in AD brains. Interestingly, loss of Pin1 causes an increase in the neuronal toxicity of Ab oligomers and NMDA. Conclusions: This study indicates that loss of Pin1 activity may induce structural changes of dendritic spines and synaptic dysfunction in AD development. Pin1 may serve as a common therapeutic target in the treatment of synaptic dysfunction and proteinmisfoldingin preclinical AD. Pin1 may also play a common pathological role in the synapses of other neurodegenerative diseases.

Particular types of beta-amyloid oligomers are formed in Alzheimer's disease-like physiopathological conditions

Background: A b oligomers are today considered as major neurotoxins involved in the pathogenesis of Alzheimer’s disease. These soluble forms of aggregated A b are sensitive to denaturation techniques used in most biochemistry protocols. Here we decided to analyse A b peptides derived from mice brain homogenates, synthetic preparations of monomeric A b 1-42 and from ADDls preparations in order to characterize and compare the nature of the oligomers present in these samples. Methods: In order to preserve the nature of oligomers formed, electrophoresis followed by western blotting were performed in non denaturating conditions. Nu1 antibody, specific of oligomeric configurations of A b was used in comparison of commonly used anti-A b (4G8, 6F3D, 6E10, FCA3542) antibodies to underline its specificity towards oligomers. Results: In synthetic preparations of monomeric A b 1-42, a range of all sizes of oligomers are formed after 15 min incubation at 37 C. In synthetic preparations of ADDLs, different types of oligomers, displaying different immunoreactivity profiles are formed depending on the final A b concentration of the samples tested. In brain homogenates of transgenic mice, we found in the extracellular soluble enriched fraction fewer types of oligomers then what was found in synthetic preparations. Among them a species corresponding to dodecameric A b has been identified.Conclusions:A b oligomers are highly unstable and are formed differently depending of the A b concentration. This degree of aggregation drives their immunoreactivity and probably their toxic properties. Interestingly oligomers formed in-vivo are very different of those formed in vitro using synthetic A b peptides suggesting the existence of partners or chaperones that stabilize and/or solubilize certain oligomeric forms in the extracellular space.

Functional correlations of intracellular Aβ peptide in transgenic mouse models

Background: Alzheimer’s disease (AD) is characterized by accumulation of extracellular amyloid plaques and the presence of intracellular neurofibrillary tangles. There is increasing evidence that Aß peptidesaccumulate also inside neurons, in addition to thewell-known parenchymal extracellular amyloid deposition and it has been hypothesized that this represents one of the earliest events triggering the neurodegenerative cascade. Transgenic mice have been proven to be valuable systems to model early intraneuronal Aß accumulation and the resulting pathological consequences. Methods: Transgenic AD mouse models (APP/PS1KI, 5XFAD, TBA2) were used to investigate if significant increases in the amount of diverse Aß species within neurons coincided with neuron loss in the respective brain regions. The presence of extracellular amyloid plaques and intraneuronal Aß accumulations was assessed by immunohistochemical stainings using antibodies against a variety of Aß peptides. Design-based stereology was used to quantify neuron numbers in transgenic mouse brains harbouring either only extracellular, or extraand intracellular Aß pathology.Results:No significant neuron loss was detected in brain regions where only extracellular plaque deposition has been detected. The presence of intracellular Aß, however, coincided with significantly reduced neuron numbers in diverse brain regions like frontal cortex, CA1 or cholinergic brain stem nuclei in APP/ PS1KI mice or cortical layer 5 in 5XFADmice. In addition, the intracellular expression of truncated Aß3-42 resulted in massive neurological impairments and abundant neuronal loss in TBA2 mice. These results lead to the assumption that extracellular Aß pathology does not significantly contribute to neuron death. Conclusions: In summary, these observations support a pivotal role of intraneuronal Aß as a major trigger in ADtypicalneurodegeneration and neuron loss, whereas plaques might represent ‘wastebins’ for toxic peptides. The link to Tau is presently not fully understood.

Amyloid deposits imaging by passive staining in mouse models of Alzheimer’s disease

A 1-42). Conclusions: Compact neuritic A plaques were abundant in the frontal cortex and absent in the cerebellum. 6-CN-BTA-1 bound to blood vessels and A 1-16/tau dual-labeled compact neuritic plaques in the frontal cortex; binding was not detected in the cerebellum. 6-CN-BTA-1 was more readily detected in A 1-40-immunoreactive (ir) plaques than in A 1-42-ir plaques. Diffuse non-neuritic A deposits were devoid of 6-CN-BTA-1 staining. Pretreatment of tissue sections with formic acid completely abolished 6-CN-BTA-1 staining. Our results demonstrate that 6-CN-BTA-1 labels preferentially compact neuritic plaques in AD brain, suggesting its affinity for binding to aggregated A . These data aid in interpreting the binding properties of the parent compound of 6-CN-BTA-1, PIB, which is currently under evaluation as an in vivo diagnostic marker of diseasespecific A plaque pathology progression in AD brains.

Neuropathology of Alzheimer’s Disease, as Seen in Fixed Tissues

The progress of in vivo imaging will in the future determine if deductions made from fixed tissue concerning the pathology of Alzheimer’s disease (AD) were correct. It will mainly enable us to apprehend directly the chronological sequence of the lesions and their duration. This paper reviews some of the data concerning the classical pathology of AD. Lesions may be categorized into Aβ peptide deposition, tau accumulation and loss of neurons and of synapses. Aβ peptide originates from the amyloid precursor protein, a transmembrane protein that is found in lipid rafts of the cellular membrane. Accumulation of flotillin-1, a raft marker, in AD indicates a disturbance of this membrane transport system in AD. Diffuse deposits of Aβ peptide within the cerebral cortex have little clinical consequence. Focal deposits are generally amyloid, i.e., Congo red positive and highly insoluble. They are usually associated with microglial activation and low-grade inflammation. Aβ peptide is initially embedded in a cell membrane and partly hydrophobic. It may actually be linked to cholesterol in the extracellular milieu, as recently suggested. Tau accumulation takes place in the cell body, the dendrites and the axons of the neurons, forming, respectively, neurofibrillary tangles, neuropil threads and coronae of senile plaques. Heiko and Eva Braak have shown that the progression of neurofibrillary pathology takes place in a stereotyped manner that appears to be correlated with the clinical symptoms. Contrary to the prediction of the amyloid cascade hypothesis, neurofibrillary pathology in the rhinal cortex and pyramidal sectors of the hippocampus most often precedes the first morphological evidence of All peptide accumulation. This observation suggests that tau and amyloid pathologies are, at the start, independent processes that secondarily interact. Data from transgenic mice support this view: neurofibrillary pathology is not observed in APP transgenic mice, except if a human mutated tau transgene has also been incorporated. Progression of the lesions is different in the entorhinal-hippocampal region and in the isocortex. In the former, tau pathology may be observed in the absence of Aβ deposition, which appears to be a relatively late phenomenon. In the isocortex, by contrast, Aβ peptide deposition is the first observable event.